ICANCER RESEARCH 46, 1943-1947, April 19861 DNA Sequence Selectivity of Guanine-N7 Alkylation by Three Antitumor Chloroethylating Agents

John A. Hartley,' Neil W. Gibson, Kurt W. Kohn, and William B. Mattes@ L.aboratory ofMolecular Pharmacology. Developmental Therapeutics Program, Division ofCancer Treatment, National Cancer institute, NiH, Bethesda, Maryland 20205

ABSTRACF (4). Both compounds are like C1EtNUs in that they produce The DNA sequenceselectivitiesof guanine-N7alkylation producedby delayed interstrand cross-linking in Mer (guanine-O@-alkyl three chloroethylating antitumor agents, I-(2-chloroethyl)-3-(cis-2-hy transferase deficient) human tumor cell lines, but not in Mer droxy)cyclohexyl-l-nitrosourea(cis-2-OH CCNU), 2-chloroethyl(meth cells, DNA-protein cross-linking in both cell types without ylsulfonyl)methanesulfonate,and 8-carhamoyl-3-(2-chloroethyl)imidazo delay, and selective killing of the Mer cells (5, 6). 15,l-dl-l,2,3,5-tetrazin-4(3H)-one (mitozolomide), were examined using We have recently investigated whether either of these agents a modificationof the Maxam and Gilbert sequencing technique. In a had a simpler alkylation chemistry than the C1EtNUs. One of region ofpBR322 DNA, 2-chloroethyl(methylsulfonyl)methanesulfonate the major alkylation routes of the C1EtNUs is the production produced approximately the same degree of alkylation at all guanines. of hydroxyethyl adducts in DNA (7). Such adducts are thought cis-2-OH CCNU, however,preferentiallyalkylated the middleguanines to have little importance in the expression ofantitumor activity in runs of three or more guanines the intensity of the reaction increased but may contribute to both the mutagenic and carcinogenic with the number of adjacent guanines in the DNA sequence. Mitozolom ide produced the same pattern of preferential alkylation but not as effects of the C1EtNUs (8, 9). In reactions with purified DNA, intensely as cis-2-OH CCNU. Three other nitrosoureas, 1-(2-chloro CIEtSoSo produced only chloroethylation products, whereas a ethyl)-3-cyclohexyl-1-nitrosourea, l-(2-fluorethyl)-3-cyclohexyl-1-nitro C1EtNU produced more hydroxyethylation than chloroethyla sourea, and l-(2-chlornethyl)-1-nifrosoureagave similar patterns of al tion (10). Mitozolomide unexpectedly produced a greater di kylation to that of cis-2-OH CCNU at pH 7.2. The ratio of 7-hydroxy versity ofproducts than did C1EtNU, including chloroethyl and ethylguanine to 7-chloroethylguanine was approximately the same follow hydroxyethyl adducts.4 Thus these three chloroethylating ing treatment of the synthetic polymersdG@•dC.and(dG.dC). with cis agents which possess a similar broad spectrum of antitumor 2-OH CCNU, indicatingthat 7-chloroethylationand 7-hydroxyethylation activity have very different patterns of alkylation. were enhanced similarly by the presence of adjacent guanines. Ethylni In the present study we have examined the base-sequence trosourea produced relatively little alkylation preference. The results suggest that the alkylating intermediates, 2-chloroethyldiazohydroxide selectivity for reaction of these compounds at guanine-N7 po and 2-hydroxyethyldiazohydroxide,tendto react preferentiallywiththose sitions of DNA by a modification of a standard DNA sequenc guanine-N7 positions the electronegativity of which is enhanced by the ing method. The major findings were that both C1EtNUs and presenceof neighboringguanines.This is consistentwith the presenceof mitozolomide exhibited differences in reaction intensities with cationic character in the alkylating centers of these intermediates. 2- different guanines. Especially striking were the disproportion Chioroethyl (methylsulfonyl)methanesulfonate and ethyldiazohydroxide ately strong reactions at runs of 3 or more guanines. C1EtSoSo would not be expected to have strong cationic character, in agreement differed from all other alkylating agents so far studied, in that with their lack of sequence selectivity. the reaction intensities for all guanines were approximately the same. INTRODUCFION C1EtNUs2 are among the most effective classes of compounds MATERIALS AND METHODS so far tested in animal tumor systems (1). These compounds, ClEtSoSo and cis-2-OH CCNU were obtained from the Drug De however, have been disappointing in the clinic (2). With the velopment Branch, National Cancer Institute. [‘4C-eihyIJClEtSoSo(9.6 acceptance that chloroethylation of DNA is the probable cause mCi/mmol) and [‘C-ethyllcis-2-OH CCNU (10.4 mCi/mmol) were of the antitumor action of the CIEtNUs and the appreciation obtained from Research Triangle Institute, NC. Mitozolomide was a of the diversity of the reactions of these compounds, other gift from Professor M. F. G. Stevens, Department of Pharmacy, Aston classes of chloroethylating compounds have been prepared University, England. T4 polynucleotide kinase, Hind III, and pBR322 which might be more confined to the desired reactions. ClEt DNA were obtained from P. L. Biochemicals, EcoRI was from New SoSo prepared by Shealy et a!. (3) is highly effective against England Biolabs, and piperidine was from Fisher. Ultra-pure urea was obtained from B. R. L. mouse tumors. Tests in the National Cancer Institute tumor Determination of Guanine-N7 Alkylation Sites in Defined DNA Se screen showed ClEtSoSo to be as good as C1EtNUs against quences. The basic technique for examining the DNA sequence speci B16 melanoma and Lewis lung carcinoma.3 Mitozolomide, ficity of alkylation is an adaptation of the Maxam and Gilbert chemical another highly effective antitumor compound developed in cleavage technique for DNA sequencing (1 1) and was originally de England which appears to be a pro-drug for chloroethyltriazen scribed by D'Andrea and Haseltine (12). Hind III digested pBR322 oimidazole carboxamide, is undergoing clinical trial in Europe DNA was 32P-labeled at its 5'-ends (1 1) and further cleaved with EcoRI. Following alkylation in 25 mrsitriethanolamine HCI:l mM EDTA, pH Received 8/19/85; revised I2/31/85; accepted 1/2/86. 7.2, at 37T for 2 h, precipitation, and washing, the DNA was treated The costs of publication of this article were defrayed in part by the payment with 1 M piperidine at 90'C for 20 mm to produce breaks specifically of page charges. This article must therefore be hereby marked advertisement in at sites of N7 guanine alkylation. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The 276 base-pair Bam HI-SaIl fragment of pBR322 5'-Iabeled at I To whom requests for reprints should be addressed, at Laboratory of Molec ular Pharmacology, Developmental Therapeutics Program, Division of Cancer the Barn HI site was isolated by preparative electrophoresis on a 0.8% Treatment, Building 37, Room 5A19, 9000 Rockville Pike, Bethesda, MD 20205. agarose gel. The 622 base-pair Hind Ill-Sall fragment of pBR322 5'- 2 The abbreviations used are: CIEtNU, l-(2-chloroethyl)-l-nitrosourea; Cl labeled at the Hind III site was isolated on a 0.6% agarose gel. EtSoSo, 2-chloroethyl (methylsulfonyl)methanesulfonate; cis-2-OH CCNU, l-(2- Alkylation was as described above. chloroethyl)-3-(cis-2-hydroxy)cyclohexyl-l-nitrosourea; mitozolomide, 8-carbam oyl-3-(2-chloroethyl)imidazo(5,I-d@-l ,2.3,5-tetrazin-4(3H)one; HPLC, high-per formance liquid chromatography. 4 J. A. Hartley, N. W. Gibson, K. W. Kohn, and W. B. Mattes, unpublished 3 J. Plowman, personal communication. data. I 943 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. SEQUENCE SPECIFICITY OF CHLOROETHYLATING AGENTS

Electrophoresis of the DNA fragments was on 0.4 mm x 90 cm x 20 and further cleaved with EcoRI, was alkylated with either of cm 6% acrylamide 5 M urea polyacrylamide gels (11). Gels were run the three agents at 37°Cfor 2 h, and the DNA was cleaved with for 3 h at 85 W (approximately 3600 V) to maintain a constant pipendine to produce breaks at sites of guanine-N7 alkylation. temperature of 50°C. Following autoradiography of the gel, relative The pattern of fragments obtained can be seen in Fig. 2. band intensities were determined by microdensitometry using a Beck man DU-8 scanning spectrophotometer with gel scanning accessory. ClEtSoSo (lanes a—c,50—150@sM)produced similar degrees of HPLC of DNA Base Adducts. [email protected]@ and (dG.dC)@ (P. L Bio alkylation at all guanines. On increasing the dose of ClEtSoSo chemicals, Inc., Milwaukee, WI) were dissolved in 0.1 M NaCI, 1 mM up to 1 mM no preferential alkylation of guanines was evident EDTA, 1 mMNaPO@,pH 7 and reacted with ‘4C-ethyl-labeleddrugat (data not shown). In contrast cis-2-OH CCNU (lanes d—f) 37°Cfor 4 h. DNA was precipitated with 95% ethanol at —20T produced occasional hot spots of alkylation. This can be seen overnight and recovered by centrifugation. Depurination was achieved in the guanines at positions 173 and 184 in the pBR322 by reaction with 0.15 Mhydrochloric acid at 95C for 30 mm. Samples sequence, which are the middle guanine in runs of three gua were neutralized with 0.3 MNaOH prior to analysis by reverse-phase nines. In the case of mitozolomide (lanes g—:)the same prefer HPLC in 5-im 4.6 mm x 25 cm C18 ultrasphere columns (Altex Ltd.) ential alkylation was observed but less intensely. on a Beckman model 344 gradient liquid chromatograph. The running Densitometric scans of the autoradiograms from Fig. 2 show buffer was 20 mMammonium formate with a 1—10%acetonitrilelinear the differing alkylation patterns produced by these three agents gradient over 30 mm; acetonitrile was then held at 10% for 10 mm followed by a linear gradient from 10—100%acetonitrileover 30 mm. at an equimolar concentration (Fig. 3). In addition to the The flow rate was maintained at I ml/min. One-mi fractions were visually striking difference observed in the two runs of three collected, 4 ml water and 10 ml Aquassure (New England Nuclear) guanines, some additional, more subtle differences can be seen, scintillation fluid were added, and the samples were counted. particularly with pairs ofguanines (e.g., guanines 163 and 164). 7-Hydroxyethylguanine and 7-chloroethylguanine standards were prepared as described previously (13, 14). a bcd • f 9 h I jk

RESULTS @ All CIEtNUs presumably generate the same alkylating inter ii mediates (chloroethyldiazohydroxide and hydroxyethyldiazo

hydroxide) and might therefore not be expected to differ in the @iIIi@@@.: I relative alkylation of various guanine sites in a DNA sequence. We have shown previously by HPLC analysis ofmodified DNA base products that ClEtSoSo produces less variety of guanine products in DNA than do the C1EtNUs (10). Mitozolomide produced a greater variety ofalkylation products than did either a CLEtNU or ClEtSoSo. Since these compounds produce dif ferent proximal reactive species, the site selectivity within DNA @ ag may differ. In the present study we have tested this hypothesis 1* a,@! in a defined region of pBR322 DNA by examination of sites of ,, ,...... guanine-N7 alkylation. @;,. The structures of cis-2-OH CCNU (a low carbamoylating nitrosourea), ClEtSoSo, and mitozolomide are shown in Fig. 1. Hind III digested pBR322 DNA, 32P-labeled at the 5'-end

;@;

@ N=O -- T 250 @ a a @TT @ :@ TAT 240 CICK@CH@—N-C-NH .@ COC :,* —. _TA 3C 230 8 — SC @@ ____GQ_@0AC@@T220 cis—2—OHCCNU(NSC-264395) 1@; ____c@@!@iT_@ @øc 210 : ASC @,. _AC a, 200 @ -.- . T 0 0 a * II II CC ClCH2CH2-0-S@-CH2-S-CH3 --. ___a______TA c1@0 @ II H . _____QC13o1___TISO 0 0 - cT' @ CIEtSOSO(NSC-338947) . OS 170

AC

NØN@.@CONH@ @ . -@-. TT C

CICH@CH2@y@¼d@ Fig. 2. Sites ofguanine N7-alkylation produced by C1EtSoSo (lanes a—c),cis 0 2-OH CCNU (lanes d—f),and mitozolomide (lanes g—i)in Hind III digested pBR322 DNA, 32P-labeled at the 5'-ends and further cleaved with EcoRl. Doses Mftozolomlde(NSC-353451) are 50iiM(a, d, andg), 100uM(b, e, and h), and l50@iM(c,f, and ,) for 2 h at 3'FT in 25 mM triethanolamine HCI:1 m@iEDTA, pH 7.2. Lanesj and k are the Fig. 1. Structures of cis-2-OH CCNU, ClEtSoSo, and mitozolomide. The formic acid purine lane and the guanine specific dimethylsulfate sequencing lanes, position ofthe ‘IC-labelisindicated(s). respectively. 1944

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. SEQUENCE SPECIFICITY OF CHLOROEThYLATING AGENTS ab c d e f gh I C1s-2-OHCCNU @ w@ @v— _ @w

LU C.)z

0 U)

,,‘ G3

M G5

GEL LENGTH (mm) Fig. 3. Densitometric scans of the guanine-N7 alkylation pattern produced by G3 cis-2-OH CCNU, mitozolomide, and CIEtSoSo at 150 MM.Scans correspond to Fig. 2, Ianesf, i, and c, respectively.

Another region containing runs of three or more guanines is present in the upper less resolved third of the gel shown in Fig. 2. Intense bands are visible in the cis-2-OH CCNU and mito zolomide alkylated samples as compared to ClEtSoSo. In order to examine this region more closely, a 276 base pair fragment @@ was prepared from this region (see “Materialsand Methods―). G Fig. 4 shows the pattern of alkylation produced by cis-2-OH- ‘@ 3 @@@ CCNU and mitozolomide in this fragment. Again the middle ,@ .@ , guanine is preferentially alkylated in the runs of three guanines resolved by the gel. In addition, and more strikingly, the middle guanines in runs of four or five guanines show strong prefer @ ential alkylation. Some preferential alkylation at the runs of @. G3 guanines can also be seen in the guanine-specific dimethylsul fate reaction (lane i). In this case, however, the preference is not confined to the middle guanine(s), and the overall increased alkylation at the runs ofguanines compared to isolated guanines @ is not as great as was seen with cis-2-OH CCNU and mitozo- ,@ a@ • G @@@ lomide. This can be seen more clearly in Table 1, which shows the average intensity of guanines in runs of 2—5guanines following alkylation. It should be noted that the intensity values presented are for the average guanine alkylation at such runs and that the reaction intensities at the individual guanines within a run can vary greatly. The intensities of alkylation by the nitrosourea and mitozolomide are enhanced by adjacent guanines in the DNA sequence. Within this same fragment, C1EtSoSo alkylated all guanines nearly equally, with an inten- Fig.4. SitesofguanineN7-alkylationproducedinthe 276base-pairBamHl slty slmllar to the Isolated guanmes alkylated by czs-2-OH SailfragmentofpBR322,5'-labeledatthe BamHlsite,showingthepreferential CCNU and mitozolomide at the same doses (data not shown). alkyiationofrunsofguaninebycis-2-OHCCNU(lanesa-a')and mitozolomide @@@@ C,. I― 1 11 I (Ianese—h).Doses,induplicate,arel5giM(Ianesa,c,e,andg)and I50@iM(Ianes ,ince @irt 5 can generate sevenu &t@yiatingspecies, lt is b,d,f, andh).Lanei is the MaxamandGilbertdimethylsulfatetreatedguanine possible that the preferentlal alkylation is due to the reaction specificsequencinglane. 1945

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. SEQUENCE SPECIFICITY OF CHLOROETHYLATING AGENTS

Table I Average intensity ofguanine N7-alkylation in runs of2—5guanines in runs of guanines might be due to enhanced chloroethylation relative to the average intensity ofa single isolated guanine or to enhanced hydroxyethylation. We have tested this using Average alkylation intensity per G the synthetic polynucleotides dG5 .dC5 and (dG .dC)@in which (average intensity of isolated Gs = I) the ratio of 7-chloroethylguanine to 7-hydroxyethylguanine Drug(G)2(G)3(G)4(G),cis-2-OH would be expected to differ if the preferential alkylation in runs l.8cMitozolomide1.5CCNUl.8 (164_216)b440 (3.29—5.61)I0.5cI of guanines observed with cis-2-OH CCNU were due to only (1.98—2.83)5.25.4ClEtSoSo1.3(1.37—1.61)2.4 (1.16-1.73)1.31.4Dimethylsulfate1.2(1.18—1.42)1.4 one of these species. The polynucleotides were alkylated with (0.77—3.55)1.4 (0.61—1.80)3.11.1 ‘4C-ethy/-labeledcis-2-OH CCNU or ClEtSoSo. Alkylated pu a Mean of five occurrences within the sequence. rines were released by treatment of the DNA with hydrochloric a Numbers in parentheses, range. acid and were separated by HPLC. The amounts of 7-chloro C Single occurrence within the sequence. ethylguanine and 7-hydroxyethylguanine produced per mg Table 2 Relativeamountsof 7-hydroxyethylguanineand7-chioroethylguaninein DNA can be seen in Table 2. In agreement with previous results dG@dC,. and (dGdC)., treated with f'4C-ethylJcis-2-OH CCNU or CIEtSoSo (10), ClEtSoSo and cis-2-OH CCNU produced approximately DNA/mM equal amounts of 7-chloroethylguanine, but ClEtSoSo pro drug duced no detectable 7-hydroxyethylguanine, which was the ma 7-OHEtG:[email protected]/mg 7-OHEtG 7-CIEtGRatio jor product with cis-2-OH CCNU. The ratio of 7-hydroxyethyl 15814.5dGD.dC.CIEtSoSo0 guanine to 7-chloroethylguanine was approximately the same 7080(dG.dC)@cis-2-OH 1 following treatment with cis-2-OH CCNU in both polymers. 3543.3(dG CCNUI 181 Three other nitrosoureas, 1-(2-chloroethyl)-3-cyclohexyl-1- dC).,CIEtSoSo0 5080 nitrosourea, 1-(2-fluoroethyl)-3-cyclohexyl-1-nitrosourea, and abcdefgh I j ki C1EtNU, known to differ in their relative production of hy droxyethylation versus haloethylation (7), produced similar pat terns of preferential guanine N7 alkylation to that produced by @G3 cis-2-OH CCNU (Fig. 5). If the reactions were carried out at pH 5 [a condition shown previously to favor the production of !P19:1 the cyclic oxadiazole intermediate (15)J instead of pH 7.2, then no significant alkylation was observed. (Dimethylsulfate pro duced alkylations under both pH conditions.) To determine if the base sequence selectivity was dependent on the nature of the alkyldiazohydroxide intermediate, alkyla tion of a fragment of DNA by C1EtNU and 1-ethylnitrosourea was compared. The resulting densitometric scan can be seen in Fig. 6. The preferential alkylation at runs of guanines observed with haloethylnitrosoureas was not seen with ethylnitrosourea.

DISCUSSION

@ — — — Guanine-N7 alkylation sites are relatively easy to localize in a DNA sequence using essentially the method of Maxam and @ — — — — Gilbert (1 1). Alkylation ofthis position facilitates the hydrolysis @‘ @- - of the guanine-deoxyribose linkage, leaving an apurinic site @ _S which can be converted into a strand break, thereby localizing the alkylation site by the usual sequence analysis procedure. In the present study three chloroethylating agents with a similar spectrum of activity were shown to possess differing DNA sequence selectivities for N7-guanine alkylation in purified DNA. cis-2-OH CCNU and mitozolomide were shown to be similar in their sequence pattern of alkylation, particularly the selective alkylation in runs of three or more guanines, although the magnitude of the selectivity was less for mitozolomide than for the C1EtNU. What chemical species generated by these drugs may give rise to the preferential alkylation at runs of guanines? Both cis 2-OH CCNU and mitozolomide can generate several alkylating Fig. 5. Sites ofguanine N7-alkylation produced in the 622 base-pair Hind III species, and it is possible that the differing patterns of alkylation Sail fragment of pBR322, 5'-labeled at the Hind III site, by four nitrosoureas: are due to the reaction of one of these species. Chloroethylation cis-2-OH CCNU (lanes c and d); l-(2-chlorocthyl)-3-cyciohexyl-l-nitrosourea (lanes e and /); l-(2-fluorocthyl)-3-cyclohexyl-I-nitrosourea (lanes g and h); and of DNA may arise, according to chemical evidence, from the CIEtNU (lanes i andj). Lanes a and b are control, untreated DNA, and lanes k alkylating species chloroethyldiazohydroxide; but in addition and Iare the Maxamand Gilbert dimethylsulfatetreatedguaninespecificsequenc both agents are capable of producing other alkylating interme ing lanes. The alkylation reaction at 500 @Mwasat either pH 5, (lanes a, c, e, g, i, and k), or pH 7.2 (lanes b, d,f, h,j, and 1). diates which can give rise to 7-hydroxyethylguanine (4, 15). The HPLC results from the alkylation of the guanine-cytosine of one of these species. We have shown previously that cis-2- polymers suggest, however, that chloroethylation and hydrox OH CCNU produces both 7-chloroethylguanine and 7-hydrox yethylation of guanine-N7 are similarly enhanced in runs of yethylguanine, whereas ClEtSoSo produced only 7-chloro guanines. In addition, C1EtNUs have been shown to decompose ethylguanine (10). The enhanced reactivity of the N7 positions by two competing mechanisms largely dependent on pH. At 1946 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. SEQUENCE SPECIFICITY OF CHLOROETHYLATING AGENTS

06 7 might exist. These “partial―chloroniumand oxonium ions shown would have some resemblance to the aziridinyl ion CHLOROETHYLNITROSOUREA species produced by nitrogen mustards. Consideration of the sequence-dependent variations in the electrostatic potential at the guanine-N7 position reveals that the site ofgreatest electro negativity is the N7 position of guanines flanked by other guanines (16). Thus an intermediate with a partial positive charge would be expected to have the greatest reaction at such sites. This hypothesis is consistent with the observation that w ethylnitrosourea and ClEtSoSo, which would be unable to Uz produce such charged intermediates, do not have strong pref erences for runs of guanines as compared to haloethylnitrosou reas. 0 (I) ETHVLN@@R0S0UREA The high chemotherapeutic activity of C1EtSoSo suggests 4 that DNA sequence specificity for reactions at guanine N7 by the nitrosoureas is not required. Alternatively, by examining only alkylation at the N7-position of guanine a possible se quence specific requirement for alkylation at the Os-position of guanine in the major groove may be overlooked. The lack of any preferential guanine alkylation with ClEtSoSo indicates that this agent may be a useful footprinting agent for the major groove. It remains to be determined to what degree the prefer ential guanine-N7 alkylation at runs of guanines is preserved GEL LENGTH (mm) in chromatin and intact cells. Fig. 6. Densitometric scans of the guanine-N7 alkylation pattern produced by CIEtNU (100 MM),and 1-ethylnitrosourea (5 mM). Filledboxes correspond to the positions of guanines within the sequence. REFERENCES e CH2—CH@---NN-0H 1. Montgomery, J. A. Chemistry and structure activity studies of the nitrosou rena. Cancer Treat. Rep., 60: 651—664,1976. \o/ 2. Prestayko, A. E., Crooke, S. T., Baker, L H., Carter, S. K., and Schein, P. Cl S. (eds.). Nitrosoureas. New York: Academic Press, Inc., 1981. 3. Shealy, Y. F., Krauth, C. A., and Laster, W. R., Jr. 2-Chloroethyl (methyl 0 sulfonyl)methanesulfonate and related (methylsulfonyl)methanesulfonates: CH2@-CH1--N=N--0H antineoplastic activity in vitro. J. Med. Chem., 27: 664—670,1984. 4. Stevens, M. F. G., Hickman, J. A. Stone, R., Gibson, N. W., Baig, G. U., 0 Lunt, E., and Newton, C. G. Antitumor imidazotetrazinones.1. Synthesis H and chemistry of 8-carbamoyl-3-(2-chloroethyl)imidazo(5,l-d@-l,2,3,5-tetra zin-4(3H)-one, a novel broad spectrum antitumor agent. J. Med. Chem., 27: Fig. 7. Structure of the proposed partial chloronium and oxonium ion inter 196—201,1984. mediates. 5. Gibson, N. W., Erickson, L C., and Kohn, K. W. DNA damage and differential cytotoxicity produced in human cells by 2-chloroethyl (methyl sulfonyl)methanesuifonate (NSC 338947), a new DNA-chloroethylating pH 7.4 the decomposition is predominantly through alkyldi agent. Cancer Res., 45: 1674—1679,1985. azohydroxides, whereas at pH 5 the decomposition is predom 6. Gibson, N. W., Hickman,J. A., and Erickson,L C. DNA cross-linkingand cytotoxicity in normal and transformed human cells treated in vitro with 8- inantly through a cyclic oxadiazole intermediate (15). In the carbamoyl-3-(2.chloroethyl)imidazo(5,l-dJ-I,2,3,5-tetrazin-4(3H).one. Can present study no significantly different alkylation was observed cer Res., 44: 1772—1775,1984. 7. Tong. W. P., Kohn, K. W., and Ludlum, D. B. Modification of DNA by at pH 7.2 between four haloethylnitrosoureas with known dif differenthaloethylnitrosoureas.CancerRes., 42: 4460—4464,1982. fering relative production of hydroxyethylation versus chloro 8. Oakberg, E. F., and Crosthwait, C. D. The effect of ethyl-, methyl-, and ethylation (7). No significant alkylation was observed at pH 5 hydroxyethyl-nitrosoureas on the mouse testis. Mutat. Res., 108: 337—344, 1983. with any nitrosourea, although dimethylsulfate did alkylate at 9. Pelfrene, A., Mirvish, S. S., and Gold, B. Briefcommunication: induction of both pHs. This suggests that the alkylation observed may be malignant bone tumors in rats by I-(2-hydroxyethyl)-1-nitrosourea. J. Natl. due to the alkyldiazohydroxide species. This is consistent with Cancer. Inst., 56:445—446,1976. 10. Gibson, N. W., Hartley, J. A., Strong, J. M., and Kohn, K. W. 2-Chloroethyl the observation that mitozolomide produces a similar pattern (methylsulfonyl)methanesulfonate (NSC-338947), a more selective DNA al of preferential alkylation, since this agent is also capable of kylating agent than the chloroethylnitrosoureas. Cancer Res., 46: 55@-557, producing 2-chloroethyldiazohydroxide and 2-hydroxyethyldi 1986. II. Maxam, A. M., and Gilbert, W. Sequencingend-labeled DNA with base azohydroxide (4). ClEtSoSo, however, cannot produce such specific chemical cleavages. Methods Enzymoi, 65: 499-560, 1980. intermediates. Furthermore, the fact that drugs such as cis-2- 12. D'Andrea, A. D., and Haseltine, W. A. Modification of DNA by aflatoxin B, creates alkali-labile lesions in DNA at positions ofguanine and adenine. OH CCNU, C1EtNU, and mitozolomide give essentially the Proc. NatE Acad. Sd. USA, 75: 4120—4124,1978. same pattern of selective alkylation argues that such a reaction 13. Gombar, C. T., Tong. W. P., and Ludlum, D. B. Mechanism ofaction of the arises from interactions of common intermediates with the nitrosoureas IV. Reactions of bis-chlomethylnitrowurea and chloroethyl cyclohexyl nitrosourea with deoxyribonucleic acid. Biochem. Pharmacol., 29: DNA rather than from interactions of the parent compound 2639—2643,1980. and DNA. 14. Tong, W. P., and Ludlum, D. B. Formation ofthe cross-linked base, diguan What features of the 2-chloroethyldiazohydroxide and 2- ylethane in DNA treated with N@'-(2-chlomethyi)-N-nitrosourea.Cancer Res., 41: 380—382,1981. hydroxyethyldiazohydroxide might contribute to the enhanced 15. Brundrett, R. B. Chemistry of nitrosoureas. Intermediacy of 4,5-dihydro reactivity at runs of guanines? While Brundrett has ruled out a 1,2,3-oxadiazole in l,3,-bis(2.chloroethyl)l-nitrosourea decomposition. J. Med. Chem., 23: 1245—1247,1980. chloronium ion as a stable species in C1EtNU chemistry (15), 16. Pullman,A., and Pullman, B. Molecularelectrostaticpotential ofthe nucleic it is still possible that the intermediate structures shown in Fig. acids. Q. Rev. Biophys., 14: 289—380,1981.

1947

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research. DNA Sequence Selectivity of Guanine-N7 Alkylation by Three Antitumor Chloroethylating Agents

John A. Hartley, Neil W. Gibson, Kurt W. Kohn, et al.

Cancer Res 1986;46:1943-1947.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/46/4_Part_2/1943

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/46/4_Part_2/1943. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1986 American Association for Cancer Research.